终末期肾病患者血浆同型半胱氨酸及其他氨基硫醇物水平变化

目的 观察终末期肾病(end-stage renal disease,ESRD)患者血液透析前后血浆同型半胱氨酸(homocysteine,Hcy)及其他氨基硫醇物的水平变化.方法 选择慢性维持血液透析患者26例,健康对照组54名.用HPLC-荧光检测法测定血浆总Hey(tHcy)、总半胱氨酸(total cysteine,tCys)、总半胱氨酰甘氨酸(total cysteinylglycine,tCysGly)、总谷胱甘肽(total Glutathione,tGSH)浓度.同时测定受检者的血脂水平和肾功能.结果 ESRD患者血液透析前血浆tHcy、tCys、tCysGly浓度分别为(16.70±3.51)μmol/L、(341.87±70.65)μmol/L和(41.33±32.95)μmol/L,明显高于健康对照组的(10.95±3.07)μmol/L、(249.76±13.18)μmol/L和(31.3±11.78)μmol/L,差异有统计学意义(t值分别为3.625、6.219和3.530,P均<0.01);ESRD患者血液透析前血浆tGSH浓度为(5.91±0.08)μmol/L,低于健康对照组的(9.33 ±2.62)μmol/L,差异有统计学意义(t=-5.404,P<0.01);血液透析后,tHcy、tCys浓度分别为(11.74±3.42)μmol/L、(272.67±64.18)μmol/L,较透析前显著降低,但不能恢复至正常水平;血液透析前后tCysGly浓度分别为(41.33±32.95)μmol/L与(44.93±13.88)μmol/L,差异无统计学意义(t=-0.758,P>0.05);tGSH浓度分别为(5.91±0.08)μmol/L与(5.93±0.38)μmol/L,差异无统计学意义(t=-0.068,P>0.05).患者血浆tHcy与tCys和Cr呈正相关(r值分别为0.753 6、0.458 2,P均<0.01),与tGSH呈负相关(r值为-0.6099,P=0.000 9),与tCysGly和血脂参数无关.结论 ESRD患者普遍存在氨基硫醇物代谢紊乱.     

The studies on the changes of nitric oxide synthase in the lung tissue of traumatic hemorrhagic shock rats under dry heat environment of desert and their relations to the secondary lung injuris北大核心CSCD

Objective: To study the pathological changes and expressions of NO and iNOS mRNA in the lung tissue of traumatic hemorrhagic shock rats under dry heat environment of desert and their relations to the lung injury. Methods: A total of 140 male SD rats were randomly (random number) ivided into the room temperature (25°C) environment traumatic hemorrhagic shock group (room temperature group) and the dry heat traumatic hemorrhagic shock groups (dry heat group, temperature 40°C, humidity 10%), respectively, and each groups was further randomly divided into 7 subgroups: the control subgroup, post shock subgroups at 0, 0.5, 1, 1.5, 2 and 3 h (re = 10 in each subgroup). The rats of control subgroup were not treated, and rats of dry heat group were placed in dry heat environment for 60 min, then anesthetized, fixed, and insertion of intravenous indwelling needles and catherization of right carotid artery, jugular vein and the right femoral artery were performed. After stabilization for 10 min, 2500 g iron wheel was used to be dropped from 30 m height and vertically hit the upper left femoral of SD rats in order to make comminuted fracture, wounds were quickly dressed after injury. Exsanguination from right femoral artery was kept until MAP maintained at (35 ± 5) mmHg, and resuscitation was carried out after continue monitoring for 60 min. After the establishment of traumatic hemorrhagic shock model in each environment, the rats were sacrificed at given intervals, and thoracotomy was performed to take broncho - alveolar lavage fluid (BALF) and lung tissue. Pathological changes of lung tissues were observed by using HE staining and NO concentration of lung tissue was detected by one-step method, and changes of the iNOS mRNA expressions were detected by using fluorescence quantitative PCR. Then t test, ANOVA and Pearson correlation analysis were used for the data analysis. Results: The pathological change in dry heat group at each interval was more severe, and pulmonary histopathological injury score was higher, and the protein exudation was more profuse compared with the room temperature group. NO concentration in lung tissue homogenate of dry heat group was higher than that of room temperature group (t = 2.472, P < 0.05), and the difference in NO level between different intervals within the dry heat group was statistically significant (F = 6.11, P < 0.01). The NO concentration in dry heat group reached its maximum at 2 h (3.35 ± 0.23) μmol/g and the peak value emerged sooner than that in room temperature group. The difference was statistically significant in overall expression of iNOS mRNA between two groups analyzed with t test (t = 3.619, P < 0.01), and there was statistically significant difference between intervals within the dry heat group (F = 12.34, P < 0.01). The values of iNOS mRNA in the dry heat group were higher than those in the room temperature group at the same given intervals, and the peak value appears at 1.5 h in dry heat group, and the room temperature group it began to increase at 2 h. The concentration of NO and the expression of iNOS mRNA were positively correlated with each other in two groups (r = 0.680, r = 0.376). The expression of iNOS mRNA and lung histopathological injury score was positively correlated in two groups (r = 0.846, r = 0.899). Conclusions When traumatic hemorrhagic shock occurred in the dry heat desert environment, the lung injury was more severe and appeared sooner than that in the room temperature environment. NO and iNOS played important roles in the secondary lung injury in the wake of traumatic hemorrhagic shock in rats under the dry heat environmengt of desert.     

全血糖化血红蛋白用常规方法测定的稳定性研究

Objective To investigate the stability of glycated hemoglobin HbA1c in whole blood sample measured by Tosoh G7, Roche/Hitachi 7170A and NycoCard READER Ⅱ under different storage conditions. Methods Three whole blood samples (EDTA anticoagulated) with different glycated hemoglobin levels and one whole blood sample (heparin anticoagulated) were collected and stored at -80 ℃, -20 ℃, 4 ℃,room temperature(15 -25 ℃), and 37 ℃ HbA1c was analyzed by each method on days 1, 2, 5, 7, 9, 14, 21,28, 35, 42, 49, 56, 63 and 70 respectively. Results The results of sample stored at -80 ℃ appear to be stable for Tesoh G7 and Roche/Hitachi 7170A method. The coefficients of variation (CV) for Tosoh G7 was 0.54%-1.22%. The CV for Roche/Hitachi 7170A was 0.86% -1.82%. When samples was detected with Tosoh G7 method, the results was consistent when the sample was stored at -20 ℃ for 14 days, 4 ℃ for 63 days, room temperature for 5 days, and 37 ℃ for less than 1 day. When samples was detected with Roche/Hitachi 7170A method, the results was consistent when the samples was stored at -20 ℃ for 21 days, 4 ℃ for 42 days, room temperature for 7 days, and 37 ℃ for less than 1 day. The NycoCard READER Ⅱ showed stability at 4 ℃ for 9 days, and room temperature for less than 1 days. Conclusions The stability of whole blood samples is dependent on different methods. Storage time under different temperatures is different.     

The changes of oxidative stress and caspase-3 in swine with traumatic hemorrhagic shock in dry-heat environment of desert北大核心CSCD

Objective To study the changes of oxidative stress and caspase-3 in swine with traumatic hemorrhagic shock in dry-heat environment of desert. Methods A total of 48 Landrace small swine were randomly ( random number) divided into 2 groups (n = 24 in each group) , and then the traumatic hemorrhagic shock was established in room temperature environment [ (25 ± 1 )°C , (35 ±5)% humidity] and in dry-heat environment [ (40. 5 ±0. 5)°C, (10 ±2)% humidity] in swine. Dry-heat environment traumatic hemorrhagic shock group ( DHS) , which was made in an artificial experiment cabin mimic the reality included swine exposed in the dry-heat environment of desert for 3 h (T0, n = 6) , T1 (50 min after shock modeling, n =6) , T2 (100 min after shock modeling, n = 6), T3 (150 min after shock modeling, n = 6). At each interval, blood sample was collected to detect urea nitrogen (BUN) and creatinine, urine sample was collected to detect neutrophil gelatinase-Associated lipoprotein (NGAL) , kidney tissue samples were collected to evaluate renal morphological and tubular scores, as well as to detect catalase ( CAT) , superoxide dismutase (SOD) and malondialdehyde (MDA). Western blot was used to detect the level of caspase-3. Traumatic hemorrhagic shock group of room temperature environment (RTS) was established and variety of assays were carried out as same as those deteced in the dry-heat environment group. Results Compared with the room temperature environment exposed group, kidney damage index, antioxidant and caspase-3 were increased in desert dry-heat environment exposed for 3 h group, but there were no statistically significant difference (P > 0.05). And from T, then on, the levels of NGAL, CAT and SOD in DHS groups were increased which were significant different from those in RTS group (P < 0. 05 or P < 0. 01). There were significant differences in BUN and creatinine at T2 between two groups ( P < 0. 05 ). At T3 , caspase-3 protein content in DHS group was significantly different from that in RTS group (P < 0. 01). Correlation analysis showed that the NGAL level was correlated with the levels to MDA (rRTS = 0. 935, rDHS = 0. 858, P <0. 01) in RTS group and DHS group. Compared with RTS group, renal tissue under light microscope showed that Bowman appeared dilated with degeneration and exfoliated epithelial cells, proximal tubule epithelial shedding, and interstitial edema in DHS group. Electron microscope showed that mitochondria became pleomorphic, endoplasmic reticulum with fold broadening. Conclusions When traumatic hemorrhagic shock happened in the desert dry-heat environment, desert dry-heat environment can aggravate kidney damage, possibly by reducing the renal tissue antioxidant enzyme content and increase renal tissue caspase-3 activity to promote renal tissue apoptosis. Antioxidant stress and apoptosis may be an important role in the prevention of the secondary kidney injury induced by traumatic hemorrhagic shock in dry-heat environment.     

严重肺挫伤患者冯·维勒布兰德因子,白介素8的动态变化及其意义

目的 通过检测严重肺挫伤患者血中冯·维勒布兰德因子(vWF),白介素8在受伤后不同时点的含量,揭示其动态变化规律及其临床价值.方法 将63例严重肺挫伤患者分成ARDS组和非ARDS组、生存组和死亡组、ISS评分<20分和ISS评分≥20分组,并引入正常对照组,使用ELISA法分别检测伤后24 h内、第3天、第5天、第7天时的血浆vWF和血清IL-8水平,观察其动态规律,并进行相关因素分析.结果 严重肺挫伤患者在各时点,血浆vWF和血清IL-8均高于正常对照组.严重肺挫伤并发ARDS组血浆vWF含量各时点逐渐升高,ARDS组在第5天、第7天血浆vWF含量显著高于非ARDS组(P<0.05).血清IL-8含量在第5天达到高峰后开始下降,两组之间在各时点差异具有统计学意义(P<0.05);死亡组血浆vWF与血清IL-8含量均显著高于生存组(P<0.05);ISS评分≥20分组血浆vWF和血清IL-8水平均在第5天达到高峰后开始回落,ISS评分<20分组在第3天达到高峰后回落.血浆vWF含量与血小板计数呈正相关,与氧合指数呈负相关.血清IL-8含量与白细胞计数、ISS评分呈正相关,与氧合指数呈负相关.结论 严重肺挫伤患者血中vWF和IL-8含量呈动态变化,反映了肺损伤的程度,并可以作为严重肺挫伤后并发ARDS的预测及预后判断指标.

Abstract：

Objective To study the clinical changes of von Willebrand factor( vWF) and interleukin-8 (IL-8) in patients with severe pulmonary contusion. Methods Sixty-three patients with severe pulmonary contusion were divided into three different classifications for the sake of comparison in different respects, namely (1) severe pulmonary contusion with ARDS group and severe pulmonary contusion without ARDS group, (2) survival group and non-survival group, and (3) ISS score <20 group and ISS scored 20 group. In addition, the normal control group was set up. The levels of plasma vWF and serum IL-8 were respectively detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) within 24 hours of injury and 1,3,5 and 7days after injury. The regularity of their changes was observed and the correlation factors were analyzed from the data. Results Compared with normal controls, the concentrations of plasma vWF and serum IL-8 were significantly increased in patients with severe pulmonary contusion in all intervals of detection. The concentrations of plasma vWF escalated gradually in severe pulmonary contusion with ARDS, and reached significantly higher levels in 5 days and 7 days after injury compared with those without ARDS group (P <0. 05). The increase in concentrations of serum IL-8 peaked in 5day after injury, and then declined. The levels of serum IL-8 were higher in patients with severe pulmonary contusion with ARDS group than those in this kind of patients without ARDS group. The levels of plasma vWF and serum IL-8 were higher in non - survival group than those in survival group (P < 0.05). The increase in levels of plasma vWF and serum IL-8 peaked and then declined in 5 days in ISS score 3:20 group, whereas it peaked and declined in 3 days after injury in ISS score < 20 group. The level of plasma vWF was positively correlated with platelets and negatively correlated with oxygenation index. The levels of serum IL-8 was positively correlated with white blood cell count and ISS score, and negatively correlated with oxygenation index. Conclusions The levels of plasma vWF and serum IL-8 were increased in patients with severe pulmonary contusion, reflecting the severity of pulmonary injury. The levels of plasma vWF and serum IL-8 were the sensitive markers for evaluating the severity of pulmonary injury and the prognosis of ARDS caused by severe pulmonary contusion.     

急性高容量血液稀释对全髋关节置换术患者血浆杀菌/通透性增加蛋白表达的影响

Objective To study the effect of acute hypervolemic hemodilution on expression of plasma bac-tericidaL/permeability-increasing protein (BPI) in patients undergoing total hip replacement. Methods Twenty ASA Ⅰ-Ⅱ patients undergoing elective total hip replacement were randomly divided into two groups (n=10 for thesia. The blood loss,blood transfusion and the time of operation were recorded. Venous blood samples were taken before anesthesia (T0) ,at the begining of operation (T1) ,30 min after operation (T2) ,and at the end of operation (T3) for determination of plasma bactericidal/permeability-increasing protein. Results The blood loss and the blood transfusion in HES group were significantly lower than that of LR group[blood loss: (560±90)ml vs (810±110) ml and blood transfusion: (200±100) ml vs (600±200) ml,t=5.562 and 5.657,P<0.001]. The plasma BPI concentrations in HES group were significantly increased at T2～T3 as compared to baseline value at T0 [(8.9±1.6)μg/L,(13.4±1.2)μg/L and (4.9±1.2)μg/L,P<0.05]. The plasma BPI concentrations in LR group were significantly increased at T2～T3 as compared to baseline value at T0 [(7.3±1.2)μg/L,(9.9±0.8) μg/L and (5.0±1.1)μg/L,P<0.05],but were lower than those in HES group (t=2.530 and 7.674,P=0.021 and 0.001 ). Conclusion Acute hypervolemic hemodilution with 200/0.5 hydroxyethyl starch can reduce blood transfusion during total hip replacement operation and also can increase the BPI level which would beneficial for the immunological function.     

急性高容量血液稀释对全髋关节置换术患者血浆杀菌/通透性增加蛋白表达的影响

Objective To study the effect of acute hypervolemic hemodilution on expression of plasma bac-tericidaL/permeability-increasing protein (BPI) in patients undergoing total hip replacement. Methods Twenty ASA Ⅰ-Ⅱ patients undergoing elective total hip replacement were randomly divided into two groups (n=10 for thesia. The blood loss,blood transfusion and the time of operation were recorded. Venous blood samples were taken before anesthesia (T0) ,at the begining of operation (T1) ,30 min after operation (T2) ,and at the end of operation (T3) for determination of plasma bactericidal/permeability-increasing protein. Results The blood loss and the blood transfusion in HES group were significantly lower than that of LR group[blood loss: (560±90)ml vs (810±110) ml and blood transfusion: (200±100) ml vs (600±200) ml,t=5.562 and 5.657,P<0.001]. The plasma BPI concentrations in HES group were significantly increased at T2～T3 as compared to baseline value at T0 [(8.9±1.6)μg/L,(13.4±1.2)μg/L and (4.9±1.2)μg/L,P<0.05]. The plasma BPI concentrations in LR group were significantly increased at T2～T3 as compared to baseline value at T0 [(7.3±1.2)μg/L,(9.9±0.8) μg/L and (5.0±1.1)μg/L,P<0.05],but were lower than those in HES group (t=2.530 and 7.674,P=0.021 and 0.001 ). Conclusion Acute hypervolemic hemodilution with 200/0.5 hydroxyethyl starch can reduce blood transfusion during total hip replacement operation and also can increase the BPI level which would beneficial for the immunological function.     

急性高容量血液稀释对全髋关节置换术患者血浆杀菌/通透性增加蛋白表达的影响

Objective To study the effect of acute hypervolemic hemodilution on expression of plasma bac-tericidaL/permeability-increasing protein (BPI) in patients undergoing total hip replacement. Methods Twenty ASA Ⅰ-Ⅱ patients undergoing elective total hip replacement were randomly divided into two groups (n=10 for thesia. The blood loss,blood transfusion and the time of operation were recorded. Venous blood samples were taken before anesthesia (T0) ,at the begining of operation (T1) ,30 min after operation (T2) ,and at the end of operation (T3) for determination of plasma bactericidal/permeability-increasing protein. Results The blood loss and the blood transfusion in HES group were significantly lower than that of LR group[blood loss: (560±90)ml vs (810±110) ml and blood transfusion: (200±100) ml vs (600±200) ml,t=5.562 and 5.657,P<0.001]. The plasma BPI concentrations in HES group were significantly increased at T2～T3 as compared to baseline value at T0 [(8.9±1.6)μg/L,(13.4±1.2)μg/L and (4.9±1.2)μg/L,P<0.05]. The plasma BPI concentrations in LR group were significantly increased at T2～T3 as compared to baseline value at T0 [(7.3±1.2)μg/L,(9.9±0.8) μg/L and (5.0±1.1)μg/L,P<0.05],but were lower than those in HES group (t=2.530 and 7.674,P=0.021 and 0.001 ). Conclusion Acute hypervolemic hemodilution with 200/0.5 hydroxyethyl starch can reduce blood transfusion during total hip replacement operation and also can increase the BPI level which would beneficial for the immunological function.     

急性高容量血液稀释对全髋关节置换术患者血浆杀菌/通透性增加蛋白表达的影响

Objective To study the effect of acute hypervolemic hemodilution on expression of plasma bac-tericidaL/permeability-increasing protein (BPI) in patients undergoing total hip replacement. Methods Twenty ASA Ⅰ-Ⅱ patients undergoing elective total hip replacement were randomly divided into two groups (n=10 for thesia. The blood loss,blood transfusion and the time of operation were recorded. Venous blood samples were taken before anesthesia (T0) ,at the begining of operation (T1) ,30 min after operation (T2) ,and at the end of operation (T3) for determination of plasma bactericidal/permeability-increasing protein. Results The blood loss and the blood transfusion in HES group were significantly lower than that of LR group[blood loss: (560±90)ml vs (810±110) ml and blood transfusion: (200±100) ml vs (600±200) ml,t=5.562 and 5.657,P<0.001]. The plasma BPI concentrations in HES group were significantly increased at T2～T3 as compared to baseline value at T0 [(8.9±1.6)μg/L,(13.4±1.2)μg/L and (4.9±1.2)μg/L,P<0.05]. The plasma BPI concentrations in LR group were significantly increased at T2～T3 as compared to baseline value at T0 [(7.3±1.2)μg/L,(9.9±0.8) μg/L and (5.0±1.1)μg/L,P<0.05],but were lower than those in HES group (t=2.530 and 7.674,P=0.021 and 0.001 ). Conclusion Acute hypervolemic hemodilution with 200/0.5 hydroxyethyl starch can reduce blood transfusion during total hip replacement operation and also can increase the BPI level which would beneficial for the immunological function.     

急性高容量血液稀释对全髋关节置换术患者血浆杀菌/通透性增加蛋白表达的影响

Objective To study the effect of acute hypervolemic hemodilution on expression of plasma bac-tericidaL/permeability-increasing protein (BPI) in patients undergoing total hip replacement. Methods Twenty ASA Ⅰ-Ⅱ patients undergoing elective total hip replacement were randomly divided into two groups (n=10 for thesia. The blood loss,blood transfusion and the time of operation were recorded. Venous blood samples were taken before anesthesia (T0) ,at the begining of operation (T1) ,30 min after operation (T2) ,and at the end of operation (T3) for determination of plasma bactericidal/permeability-increasing protein. Results The blood loss and the blood transfusion in HES group were significantly lower than that of LR group[blood loss: (560±90)ml vs (810±110) ml and blood transfusion: (200±100) ml vs (600±200) ml,t=5.562 and 5.657,P<0.001]. The plasma BPI concentrations in HES group were significantly increased at T2～T3 as compared to baseline value at T0 [(8.9±1.6)μg/L,(13.4±1.2)μg/L and (4.9±1.2)μg/L,P<0.05]. The plasma BPI concentrations in LR group were significantly increased at T2～T3 as compared to baseline value at T0 [(7.3±1.2)μg/L,(9.9±0.8) μg/L and (5.0±1.1)μg/L,P<0.05],but were lower than those in HES group (t=2.530 and 7.674,P=0.021 and 0.001 ). Conclusion Acute hypervolemic hemodilution with 200/0.5 hydroxyethyl starch can reduce blood transfusion during total hip replacement operation and also can increase the BPI level which would beneficial for the immunological function.     

Novel approach for the determination of the redox status of homocysteine and other aminothiols in plasma from healthy subjects and patients with ischemic stroke

BACKGROUND: Plasma "redox" status can be assessed by measurements of reduced (r)-, free (f)-, oxidized (ox)-, and protein-bound (b)-homocysteine (Hcy) plus the related aminothiols cysteine, cysteinylglycine (CysGly), and glutathione (GSH), but sample collection has been complex. The redox status has not been determined in ischemic stroke patients and may provide increased understanding of its role in pathogenesis. We wished to examine the feasibility of this measurement in samples collected in readily available acidic sodium citrate. METHODS: We measured aminothiols and their stability in stabilized protein-free filtrate using acidic sodium citrate (BioPool Stabilyte, pH 4.3) vs EDTA whole blood. Before analysis, plasma samples were also ultrafiltered to obtain a protein-free filtrate. The concentrations of total Hcy (tHcy), fHcy, and rHcy and their related aminothiols, cysteine, cysteinylglycine, and glutathione were simultaneously determined on acidic sodium-citrated blood using reversed-phase HPLC with fluorescence detection. Bound and oxidized aminothiols were calculated by difference using the concentrations of the total, free, and reduced fractions. Using this approach, we compared the redox status in newly diagnosed ischemic stroke patients (n = 20) and healthy age- and sex-matched subjects (n = 20). RESULTS: tHcy, tCys, tCysGly, and tGSH concentrations in whole blood with Stabilyte were stable for 8 h; the reduced fraction of each aminothiol was stable for 4 h. Recovery in the protein-free filtrate was 90-100% for all reduced thiols in acidified sodium-citrated blood. Patients with ischemic stroke had higher plasma tHcy, fHcy, bHcy, rHcy, and oxHcy (P <0.0005) and higher plasma t-, f-, r-, and oxCys (P <0.05). t-, b-, and rCysGly concentrations were lower in the stroke patients (P <0.05), as were t-, b-, and oxGSH (P <0.005). CONCLUSIONS: Collection of blood in acidic sodium citrate (BioPool Stabilyte) permits the determination of the redox status of Hcy and its related aminothiols, which may add to the understanding of their relationship to the etiology of cerebrovascular disease.     

Effects of temperature on stability of blood homocysteine in collection tubes containing 3-deazaadenosine

BACKGROUND: The accuracy of homocysteine (Hcy) results is currently compromised by the requirement to separate the plasma within 1 h of sample collection. We studied the effect of temperature on the stability of plasma Hcy over a 72-h time course in blood collected into evacuated tubes containing either EDTA alone or both EDTA and 3-deazaadenosine (3DA). METHODS: We recruited 100 volunteers, including both diseased and healthy individuals with a range of baseline plasma Hcy values, from two centers. Blood samples were collected into tubes containing EDTA, and EDTA plus 3DA and stored at ambient temperature (20-25 degrees C) or refrigerated (2-8 degrees C). Aliquots of blood were centrifuged at various times up to 72 h, the plasma was removed, and Hcy was measured by HPLC. RESULTS: Plasma Hcy measurement covering the sample collection and storage conditions during the whole time course was possible on samples from 59 of those recruited. One-way ANOVA for repeated measures within subjects revealed that only samples that were collected into tubes containing EDTA plus 3DA and stored refrigerated were stable over 72 h (P = 0.2761). CONCLUSIONS: A combination of 3DA and storage at 2-8 degrees C will allow collection of samples for plasma Hcy measurement outside of the hospital setting and wider population screening.     

Better stability of total homocysteine measurement with sodium fluoride than with EDTA

The total homocysteine (tHcy) plasma concentration increases 10% per hour when whole blood is collected on ethylene diamine tetraacetate (EDTA) and stored at room temperature. The aim of this study was to investigate the stability of tHcy plasma concentration during 24 hours of storage at room temperature in two different collection tubes: EDTA and sodium fluoride (NaF). The evolution of tHcy plasma concentration was also compared in two different populations: healthy individuals (controls) and patients with end-stage renal failure, known to have increased plasma tHcy concentrations. Plasma was separated from erythrocytes at 0, 2, 6 and 24 hours. tHcy was measured with a competitive immunoassay on Immulite 2000 (Diagnostic Products Corporation). Plasma tHcy concentration started to rise significantly on EDTA after two hours of storage in patients and controls in comparison to baseline (defined as time: 0 hour). It remained stable on NaF during the first two hours and started to rise significantly after six hours of storage for both populations. In conclusion, NaF tubes should be preferred to EDTA tubes for tHcy determination in routine clinical chemistry laboratories.     

Measurement of homocysteine and other aminothiols in plasma: advantages of using tris(2-carboxyethyl)phosphine as reductant compared with tri-n-butylphosphine

BACKGROUND: Aminothiols have been implicated in the pathogenesis of arteriosclerosis, and reliable methods are needed to determine their concentrations in body fluids. We present a comparison of two analytical methods and focus on the reduction of low-molecular weight and protein-mixed disulfides of homocysteine, cysteine, cysteinyl-glycine, and glutathione. METHODS: The plasma total aminothiol profile was determined by HPLC with fluorescence detection after derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate. Disulfides and protein-bound aminothiols were reduced by either tri-n-butylphosphine (the TBP method) or tris(2-carboxyethyl)phosphine (the TCEP method); the effects of temperature, time of reduction, and concentration of reductants were evaluated. RESULTS: The intraassay imprecision (CV) was <3% for all aminothiols using both methods. The interassay CVs for total cysteine (tCys), total cysteinyl-glycine (tCys-Gly), and total homocysteine (tHcy) were <4% and <8% for the TCEP and TBP methods, respectively, whereas for total glutathione (tGSH) the interassay CV was >12% for both methods. Deming regression and Bland-Altman difference plots showed positive biases for total aminothiol concentrations determined by the TCEP method relative to the TBP method. The mean proportional biases were 65%, 27%, 6%, and 60% for tCys, tCys-Gly, tHcy, and tGSH, respectively. The calculated concentrations of total aminothiols by the TCEP method were less influenced by changes in temperature and concentration of reducing agent or by calibrator matrix. CONCLUSIONS: The agreement between the TCEP and TBP methods was considerably lower for the determination of tCys, tCys-Gly, and tGSH than for tHcy. For total-aminothiol determination, the TCEP method yields better reproducibility and is more robust than the TBP method.     

Can fluoride-oxalate and sodium citrate stabilise homocysteine levels after blood collection?

The concentration of homocysteine (Hcy) rises rapidly after the collection of blood. This feature requires blood to be collected into the anticoagulants EDTA or heparin and the plasma to then be immediately separated; alternatively, the blood may be kept on ice and centrifuged within 1 hour. The use of chemical preservatives has been proposed as a means of stabilising Hcy levels in whole blood after collection. The objective of this study was to determine whether the commonly available fluoride-oxalate (Fl-Ox) and sodium citrate (Na-Cit) containers could stabilise Hcy levels in blood. Our results showed that when blood was collected into potassium EDTA (K-EDTA) tubes, Hcy levels rose from initial levels, on standing at room temperature (approximately 25 degrees C), by an average of 21% after 3 hours and 32% after 5 hours. The initial Hcy levels of blood collected into Fl-Ox and Na-Cit containers, however, were lower, at averages of 89% and 91%, respectively, compared to that of the same samples when collected into K-EDTA tubes. Hcy in these samples subsequently rose on standing, and after 5 hours was, on the average, 10 and 13% higher, respectively, compared with the initial levels in K-EDTA tubes. We conclude that Fl-Ox and Na-Cit do not stabilise Hcy in blood after collection and should not be used as preservatives.     

The influence of smoking on plasma homocysteine and cysteine levels in passive and active smokers.

Total plasma homocysteine (tHcy) and cysteine (tCys) levels are associated with cardiovascular diseases. One of the determinants that influence their levels is cigarette smoking. The aim of this study was to determine the relationship between plasma levels of both amino acids and urinary cotinine concentration as a reliable biomarker of tobacco smoke exposure. One hundred and seventeen volunteers (61 women and 56 men) aged 19-60 years (mean 40.3 +/- 11.0) were included in the study. The study subjects were qualified into non-smokers, passive smokers and active smokers based upon the urinary cotinine concentration. In each particular group, plasma tHcy and tCys levels were measured and evaluated in the whole population and separately in women and men. Statistically insignificant differences in plasma tHcy and tCys levels in the whole group of passive smokers in comparison with non-smokers were observed (11.47 vs. 10.94 micromol/l, p=0.414, and 253.0 vs. 266.9 micromol/l, p=0.163, respectively). However, statistically significant differences in plasma tHcy levels (13.29 vs. 10.94 micromol/l, p=0.011) and in plasma tCys levels (218.2 vs. 266.9 micromol/l, p<0.001) were found in the whole group of active smokers compared with non-smokers. The Pearson's coefficient (r) for the correlation between plasma tHcy level and urinary cotinine concentration was r=0.630 (p<0.001) in the whole group of active smokers and r=0.480 (p=0.003) in the whole group of passive smokers. The correlation between plasma tCys level and urinary cotinine concentration in both study groups was insignificant. Similar results were obtained when calculated separately for men and women. The results suggest that cigarette smoking is a strong determinant of plasma tHcy level, but it is not a determinant of plasma tCys level.     

Serum cystatin C in renal transplant patients

BACKGROUND: Waiting temperature before centrifugation and anticoagulants used, markedly effect total homocysteine concentrations. The aim of this study was to investigate the effect of different anticoagulants and temperature on plasma homocysteine levels. METHODS: We studied total homocysteine concentrations in 23 healthy subjects. Blood was drawn in K(3)EDTA, sodium citrate- or sodium fluoride-containing tubes, and kept at 0 degrees C or 22 degrees C for 3 h. Total homocysteine measurements were performed with fluorescence polarization immunoassay (FPIA) method. We compared all results with baseline EDTA values (samples put on crushed ice and centrifuged immediately) recommended in literature for reference handling. RESULTS: At 22 degrees C, the tubes containing sodium citrate and sodium fluoride showed significantly higher total homocysteine concentrations than their respective baseline values (p=0.000). However, sodium fluoride tubes were not significantly different than baseline EDTA levels. Waiting 3 h at 0 degrees C did not effect sodium citrate and EDTA plasma total homocysteine concentrations when compared to baseline EDTA, but sodium fluoride-containing plasma levels were significantly decreased (p=0.000). CONCLUSIONS: According to our results, the most available and practical temperature and anticoagulant for total homocysteine determination is sodium fluoride at room temperature up to 3 h.     

血浆与血清同型半胱氨酸在不同检测条件下比较及稳定性研究

目的分析同型半胱氨酸(Hcy)水平在血浆与血清中的差异及放置不同时间检测的稳定性。方法分别以EDTA-K2抗凝管和分离胶管收集42例健康体检人员血液,于即刻及放置3h后分离血浆及血清。选EDTA-K2抗凝管立即离心分离血浆后于室温放置1h、3h、6h及2-8℃24h;分离胶管放置30min待血液凝固后离心分离血清于室温放置1h、3h、6h及2-8℃24h,使用直接化学发光法检测同型半胱氨酸浓度。结果分离胶管取血放置30min后离心分离血清同型半胱氨酸浓度较即刻分离的血浆同型半胱氨酸浓度高13.24%,结果差异有统计学意义(P<0.05);取血放置3h后离心检测血清或血浆同型半胱氨酸浓度明显高于取血后即刻离心检测的同型半胱氨酸浓度,两者相差12.47%和12.56%,结果差异有统计学意义(P<0.05)。分离后血浆与血清在室温放置1h、3h、6h及2-8℃24h检测同型半胱氨酸浓度,结果差异均无统计学意义(P>0.05)。结论血清同型半胱氨酸水平明显高于血浆,分离血清或血浆前标本放置时间对检测同型半胱氨酸浓度有影响,分离后血浆或血清在2-8℃保存,24h内同型半胱氨酸检测结果差异无统计学意义。在临床工作中,应尽可能快速分离血浆标本进行同型半胱氨酸检测或分别设立血浆与血清同型半胱氨酸浓度的正常参考范围。     

Stability of YKL-40 concentration in blood samples

The stability of YKL-40, a mammalian member of the family of 18 glycosyl-hydrolases, in blood samples handled under different temperatures and different time intervals before centrifugation was studied in paired serum and plasma samples from 25 healthy premenopausal Danish women. Significant elevations of YKL-40 were found in 8 paired serum samples left on the clot for more than 3 h at room temperature compared to paired serum samples left on the clot for 3 h or less. Significant elevations of YKL-40 were found in 8 paired plasma (EDTA) samples left on the blood cells for more than 8 h at room temperature compared to paired plasma (EDTA) samples left on the blood cells for 8 h or less. No elevations were found in YKL-40 levels in serum samples left on the clot at 4°C for 24 h or in plasma (EDTA) samples left on the blood cells for 72 h before centrifugation. Significantly lower concentrations of YKL-40 were measured in plasma (EDTA) compared with paired serum samples with a serum/plasma ratio of 1.4 in samples left on the clot or on blood cells at 4°C for up to 24 h. Repetitive freezing and thawing had no significant effect on the measured YKL-40 concentrations. In conclusion, we have shown that YKL-40 is very dependent on the handling procedures. All the blood samples must be processed into plasma (EDTA) within 8 h at room temperature or into serum in less than 3 h at room temperature. If this is not possible, the blood samples must be stored at 4°C until processed.